The integrity of the genetic information encoded by DNA is essential to all organisms, yet the reactive bases of DNA are continuously subjected to chemical modification from endogenous and exogenous sources. To counteract this inevitable damage, the cellular machinery includes DNA repair systems. Damaged bases in DNA are repaired via base excision repair (BER), initiated by a damage-specific DNA glycosylase. These enzymes find lesions within the vast genomic DNA, and hydrolyze a generally stable bond to release the damaged base, producing apurinic/apyrimidinic (AP) DNA. DNA glycosylases typically bind the cytotoxic AP DNA product tightly until displaced by an AP endonuclease to continue BER. Although some enzymes recognize a single lesion;e.g., uracil DNA glycosylase is exquisitely specific for uracil, others recognize multiple lesions and/or mismatched bases. Two human enzymes recognize G/T mispairs, and other mutagenic lesions, specifically at CpG sites;methyl-binding domain IV (MBD4) and thymine DNA glycosylase (TDG). The long-term goal of this research is to understand how these enzymes recognize complex and multiple forms of damage and yet exclude normal base pairs, how they catalyze the hydrolysis of a generally stable bond, and how the AP DNA product is transferred from the DNA glycosylase to the AP endonuclease. The focus of this proposal is to determine how TDG obtains its specificity for G/T mispairs and other lesions, and its specificity against normal GC pairs, and how human AP endonuclease (APE1) stimulates the release of AP DNA from TDG. Towards this end, we will employ a multidisciplinary approach including structural, biophysical, and biochemical methods. The specific aims are to (i) determine the NMR structure of the TDG catalytic domain (TDGc), (ii) characterize the TDG reaction mechanism using transient and steady-state kinetics, and equilibrium binding experiments, (iii) discover the chemical basis of the recognition of multiple substrates and the rejection of GC by TDG, (iv) elucidate the mechanism of AP DNA transfer between TDG and APE1, and (v) determine the NMR structure of a binary TDGc-DNA substrate analog complex to reveal the structural basis of TDG specificity. Given the mutagenic and cytotoxic effects of damage occurring at CpG sites in human genomic DNA, the proposed structural and mechanistic studies of TDG may have significant implications for ageing, and diseases including cancer.